J Ginseng Res Vol. 37, No. 3, 315-323 (2013) http://dx.doi.org/10.5142/jgr.2013.37.315
E-mail: dkrhee@skku.edu
Tel: +82-31-290-7707, Fax: +82-31-290-7727
*Corresponding author
INTRODUCTION
The antistress effects of ginseng are well known, and its active ingredients have been identified as ginsenosides [1,2]. Immobilization (IMO) stress-induced polyamine levels were decreased by ginsenosides Rb1 and Rg3, and by ginseng total saponin in mice brains [3]. Rb1, the main ingredient of ginseng, has a neuroprotective effect against glutamate-induced neurotoxicity [4,5], ischemia [6], neurodegeneration [7], and seizures [8]. Rg3 in fer- mented red-ginseng has anti-stress [4] and neuroprotec- tive effects [9].
Antistress effect of red ginseng in brain cells is mediated by TACE repression via PADI4
Eun-Hye Kim
1, In-Hye Kim
1, Jung-Ah Ha
1, Kwang-Tae Choi
2, Suhkneung Pyo
1, and Dong-Kwon Rhee
1*1School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Korea
2Hanbang Bio Research Institute, Kyung Hee University, Suwon 443-470, Korea
Ginseng is known to have antistress effects. Previously, red ginseng (RG) was shown to repress stress-induced peptidyl arginine deiminase type IV (PADI4) via estrogen receptor β (ERβ) in the brain, thus inhibiting brain cell apoptosis. Moreover, tumor necrosis factor (TNF)-α plays a critical role in immobilization (IMO) stress. However, the signaling pathway of RG-mediated repressesion of inflammation is not completely understood. In this study, we determined how RG modulated gene expression in stressed brain cells. Since secretion of TNF-α is modulated via TNF-α converting enzyme (TACE) and nuclear factor (NF)-κB, we examined the inflammatory pathway in stressed brain cells. Immunohistochemistry revealed that TACE was induced by IMO stress, but RG repressed TACE induction. Moreover, PADI4 siRNA repressed TACE expression compared to the mock transfected control suggesting that PADI4 was required for TACE expression. A reporter assay also revealed that H2O2 oxidative stress induced NF-κB in neuroblastoma SK-N-SH cells, however, RG pretreatment repressed NF-κB induction. These findings were supported by significant induction of nitric oxide and reactive oxygen species (ROS) by oxidative stress, which could be repressed by RG administration. Taken together, RG appeared to repress stress-induced PADI4 via TACE and NF-κB in brain cells thus preventing production of ROS and subsequently protecting brain cells from apoptosis.
Keywords: Panax ginseng, Oxidative stress, Peptidyl arginine deiminase type IV, Tumor necrosis factor-α converting enzyme, Nuclear factor-κB
This is an Open Access article distributed under the terms of the Cre- ative Commons Attribution Non-Commercial License (http://creativecom- mons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received 01 Feb. 2013, Revised 21 Mar. 2013, Accepted 30 Apr. 2013
The structure of ginsenoside is similar to that of ste- roids [10]. Ginsenoside Rh1 and Rb1 showed estrogen receptor (ER) activation and ER-dependent action, respectively, in the mammary gland and uterus [10].
Ginseng also activates ER in breast cancer cells in vitro but not in vivo [11]. Although ER was activated in en- dothelial cells by protopanaxadiol and protopanaxatriol ginsenosides in vitro [12], ER is not regulated by Rh2 in brain astrocytes [13]. These results indicate that the effect of ginseng on the brain should be further investigated.
Edited by Hyeyoung Min, Chung-Ang University, Korea
Membrane lipids in the brain can undergo oxidative damage upon physical stress such as a cold swim, elec- tric foot shock, and IMO [14]. However, investigation of the anti-stress effects of ginseng and ginsenosides has been focused on biochemical parameters such as plasma interleukin (IL)-6 [15], plasma cholesterol, glucose, se- rum corticosterone [16], in vitro free radical scavenging activity [17], proinflammatory cytokines (tumor necrosis factor [TNF]-α, IL-1β, and IL-6) [18], and malondialde- hyde levels [19]. Rg1 also prevented glutathione reduc- tion and superoxide dismutase activation induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the brains of mice [20]. Moreover, ginseng increases the release of cytokines and the expression of toll-like receptor 4 [21]. Our previous study showed that IMO as well as in vitro oxidative stressors such as H2O2, acryl- amide, and an endoplasmic reticulum stressor tunica- mycin significantly induced peptidyl arginine deiminase type IV (PADI4) gene. Moreover, pretreatment with red ginseng (RG, steamed and dried ginseng) derepressed ERβ, which subsequently represses induction of PADI4.
Consistently, RG pretreatment inhibits the production of cyclooxygenase 2 and malondialdehyde demonstrating that RG protects the brain from cell death by repressing PADI4 via ERβ stimulation [22]. However, the mecha- nism whereby RG affects gene expression of TNF-α and downstream nuclear factor (NF)-κB remains unknown.
Here we found that RG repressed PADI4 via TNF-α convertase (TACE) and NF-κB in brain cells, thus pre- venting production of reactive oxygen species (ROS) and subsequently protecting brain cells from apoptosis.
MATERIALS AND METHODS
Cells, animals, immobilization stress, red ginseng, and treatment
Human neuroblastoma SK-N-SH cells (ATCC HTB- 11; American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 (Lonza, Walkersville, MD, USA) media containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (10,000 U penicil- lin/mL, 10,000 mg streptomycin/mL), 1 mM HEPES (4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid), 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L bi- carbonate, 2 mM L-glutamine at 37°C, and 5% CO2. Rat neuroglioma C6 (ATCC CCL-107, American Type Culture Collection) was cultured in Dulbecco’s modi- fied Eagle medium media containing 10% FBS and 2%
penicillin-streptomycin. RG stock was prepared at 10 mg/mL in phosphate buffer saline (PBS, pH 7.4), diluted
with RPMI 1640 media containing 10% FBS and 2%
penicillin-streptomycin to 1 mg/mL just prior to use and sterilized by filtration with a 0.22 mm bottle top filter (Nalgene, Rochester, NY, USA). Male ICR mice (20 to 25 g) were housed in a temperature-controlled environ- ment (temperature 21±2°C, humidity 60±10%) and 12- hour dark: 12-hour light cycle under conditions where food and water were freely available. All experiments conformed to the animal care guidelines of the Korean Academy of Medical Sciences, and all efforts were made to minimize animal suffering. Stress procedures were approved and monitored by the ethical committee of Sungkyunkwan University. For stress experiments, mice were immobilized for 30 or 45 min in a tightly fit- ted, 50 mL conical tube. At the end of the stress period, the mice were sacrificed by cervical dislocation, and the brain was rapidly removed and frozen. Animals that were set free in their home cage in the absence of any stress- ors served as controls (the normal control group). Mice were divided into three groups: non-treated (no stress), stress + no treat, and stress + RG. The non-treated group was used as a control. The stress + no treat group was administrated only PBS. Lastly, the stress + RG group was administered RG orally (RG extract; Korea Ginseng Corporation, Daejeon, Korea) twice a day for one week.
The mice began fasting on the evening of the 7th day and were sacrificed 3 h after the last administration of RG on the morning of the 8th day.
For in vitro experiments, a filtered RG solution was further diluted with RPMI 1640 media to 1 mg/mL prior to RG treatment. In addition, cells were treated with RG for 48 h.
Determination of nitric oxide and reactive oxygen species
For ROS determination, 2×105 SK-N-SH cells were cultured overnight, and treated with 1 mg/mL of RG for 48 h or RPMI 1640 media alone (control). After treat- ment with 0.5 mM H2O2 for 20 min, cells were washed with PBS, and the intracellular accumulation of ROS was measured using the fluorescent probe H2DCFDA. At the end of the treatments, cells were loaded with 20 μM H2DCFDA and incubated at 37°C for 30 min in the dark.
Cells were then collected and resuspended in PBS. The fluorescence was measured immediately by flow cytom- etry (Biorad, Hercules, CA, USA).
For nitric oxide (NO) determination, C6 cells were pretreated with 0.5 mg/mL of RG for 24 h, and then 1 μg/mL of lipopolysaccharides (LPS; Sigma, St. Louis, MO, USA) was added. The amount of NO2- in cell su-
pernatants was measured spectrophotometrically by the Griess reaction. Samples were supplemented with 276 mU of nitrate reductase and 40 μM NADPH and then al- lowed to react with the Griess reagent (aqueous solution of 1% sulfanylamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4) in order to form a stable chromophore absorbing at a wavelength of 546 nm.
Purification of total RNA
Total RNA from mouse brain or tissue cultured cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA quality was measured spectrophoto- metrically.
Reverse transcriptase polymerase chain reaction Total RNA was reversely transcribed to complemen- tary DNA by M-MLV reverse transcriptase (RT) (Rex- Gene Biotech, Ochang, Korea). All polymerase chain reaction (PCR) primer pairs were designed for mRNA sequencing within 200 bp; p53, 5’-CTGAGG TTGGC- TCTGACTGTACCAC-CATCC3’ (forward) and 5’CTCATTCAGCTCTCGGAACATCTCGA-AGCG3’
(reverse); b-actin, 5’-TGG AAT CCT GTG GCA TCC ATG AAA-3’ (forward) and 5’-TAA AAC GCA GCT CAG TAA CAG TCC G-3’ (reverse). Relative quantifi- cation of select mRNA was performed on 20 µl of cDNA using StepOne (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The RT PCR conditions were optimized to comprise an ini- tial denaturation step of 10 min at 95°C, followed by 35 cycles of 95°C for 10 s, 55°C for 15 s and 72°C for 20 s.
Statistical analysis was by analysis of variance between groups (ANOVA).
Western blot analysis
Cells were collected by centrifugation after wash- ing with PBS, and resuspended in 50 mM Tris-Cl pH 7.4, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride and 1X protease cocktail inhibitor. Cells were lysed by sonication and cell lysates were harvested after centrifugation at 12,000 rpm, 4°C for 15 min. The amount of protein was determined by Bradford assay and 30 to 40 mg of protein was used for Western blot. Proteins separated by 10% or 15% SDS- polyacrylamide gel electrophoresis were electroblotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was then incubated
in blocking buffer (7% skim milk, in TPBS [PBS, 0.1%
Tween 20]) at room temperature on a shaking incuba- tor for more than 1 h. After washing 3 times with TPBS, the membrane was incubated in a primary antibody (1:1,000) for 1 h at room temperature with primary an- tibodies PADI4, TACE (Abcam, Cambridge, England), Bcl-2, caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (Sigma), or p65 NF-κB (Cell Signal- ing, Beverly, MA, USA). After extensive washing, the membrane was incubated with a secondary antibody (horseradish peroxidase-conjugated anti-IgG antibody, 1:5,000 or 1:10,000 anti-rabbit, anti-mouse; Sigma) for 1 h. Bands were detected with Power Optic-ECL western blotting detection reagent (iNtRON Biotech, Seongnam, Korea).
Immunohistochemistry
In order to determine the level of TACE, immunohis- tochemistry was performed on mouse formalin-fixed, paraffin-embedded 3-μm-thick sections with Discovery XT (Ventana Medical Systems, Tucson, AZ, USA).
TACE antibody (Abcam) was used at a 1:100 dilution.
For immunofluorescence, anti-red fluorescent protein antibody (Rockland, Gilbertsville, PA, USA) was used at 1:200 followed by a Alexa Fluor 488 anti-rabbit second- ary antibody (Invitrogen) according to the manufacturer’s protocol.
Transfection
To knock down PADI4 expression by siRNA, 100 nM of siRNA (Genolution Phamaceuticals, Seoul, Korea) was added to 250 mL of serum-free RPMI 1640 media without antibiotics, to which 5 mL of transfection reagent (TransIT-TKO; Mirus, Madison, WI, USA) had been added and incubated for 20 min at room temperature.
SK-N-SH cells (3×105 cells/well) were cultured in 6-well plates overnight, washed once with serum-free media without antibiotics, and replaced with 1 mL of serum-free media containing antibiotics. Subsequently, the transfec- tion reagent containing siPADI4 was added to the cell culture and incubated for 24 h. As a control, cells were transfected with mock siRNA. After 24 h incubation, siRNA was removed and replaced with new complete media containing 1 mg/mL RG (cells in RPMI 1640 me- dia alone were used as a control) for 48 h. Subsequently, cells were washed with PBS twice, and exposed to 0.5 mM H2O2 for 2 h followed by washing and lysis.
Luciferase reporter gene assay
NF-κB was determined by the luciferase reporter gene
assay according to the manufacturer’s protocol. Briefly, SK-N-SH cells were seeded in a 12-well plate with 1×105 cells per well in 1 mL of RPMI 640 media. Then, cells were transfected with 1 μg of NF-κB plasmid (pGL3- NFκB) with the Renilla luciferase reporter mRNA. After 24 h, cells were treated with RG for another 24 h fol- lowed by treatment with 0.1 mM H2O2 for 4 h. Then the cells were collected in a passive lysis buffer (Promega, Madison, WI, USA), and the supernatant was used for determination of luciferase activity with the reporter as- say system (Promega).
Statistical analysis
Data were analyzed by ANOVA followed by Dunett’s t-test for comparisons between groups. Significance was accepted when p<0.05 (*p<0.05, **p<0.01, and
***p<0.001). Data are expressed as median±standard de- viation for three to five independent experiments.
RESULTS
Reversion of cell death-associated gene expres- sion by red ginseng
Previously, we demonstrated that RG represses PADI4 expression as well as cell death, and induced Bcl-2, an anti-apoptotic factor, in vivo. Moreover, RG repressed
active p53 expression after oxidative stress in vitro [22].
However, other apoptosis-related factors such as cas- pase-3 and p53 were not examined in vivo. To confirm the anti-apoptotic nature of RG, we investigated these two markers after IMO stress. When mice were sub- jected to IMO stress, the level of p53 mRNA was sig- nificantly repressed. However, RG pretreatment reversed this repression back to normal (Fig. 1A). To corroborate anti-apoptosis at the protein level, we determined the ex- pression levels of representative apoptotic/anti-apoptotic markers caspase-3 and Bcl-2 by Western blot. Consis- tently, RG pretreatment increased Bcl-2 and repressed caspase-3 (Fig. 1B) indicating that RG could reverse IMO-induced apoptosis.
Repression of tumor necrosis factor-α convertase by red ginseng
System biology analyses previously showed that IMO stress up-regulated immuneresponse-induced genes, and TNF-α was found at the center of the network [22].
Moreover, TNF-α was significantly induced by IMO stress but RG administration reversed TNF-α induction [22]. Normally, TNF-α is bound on the cell membrane in an inactive form, and is cleaved by a TACE (synonyms:
ADAM-17 [a disintegrin and metalloproteinase 17], CD156b, cSVP and MGC71942) to be released in an ac-
Fig. 1. Decrease of cell death-associated gene expression by red ginseng (RG) in vivo. (A) Mice (3 mice/group) were administered RG for 1 wk followed by immobilization (IMO) stress for 45 min. RNA was purified from brain, and two to three samples from each group were used for reverse transcriptase polymerase chain reaction analyses. (B) Mice (5 mice/group) were administered RG for 1 wk followed by IMO stress for 45 min. Five samples from each group were mixed and used for Western blot analyses. Experiments were performed twice independently, and rep- resentative data from two experiments are shown. PBS, phosphate buffer saline. *p<0.05 and **p<0.01.
tive soluble form [23]. To further elucidate the function of RG in TNF-α induction, the TACE level in the brains of IMO-stressed mice was determined by immunohisto- chemistry. TACE was induced by IMO specifically in the medulla and the spinal cord (Fig. 2). In addition, LPS, a well-known inducer of TNF-α, also induced TACE.
However, pre-administration of RG repressed induction of TNF-α (Fig. 2) indicating that TNF-α induction in the IMO-stressed brain could be ascribed to TACE activa- tion.
TNF-α and ROS activate c-Jun N-terminal kinases (JNK) [24], and JNK further activates p53 as well as other apoptotic factors [25]. Therefore, to further inves- tigate how RG affected cell death after PADI4 induction, SK-N-SH cells were transfected with siPADI4 prior to H2O2 stress, and expression of TACE was determined by Western blot. As a control, siPADI4 was shown to re- press PADI4 expression (Fig. 3). Interestingly, expression of TACE was not affected by siPADI4 in the non-RG- treated stressed condition. However, RG pretreatment repressed expression of TACE compared to the mock transfected control under oxidative stress conditions (Fig.
3). These results indicated that oxidative stress induced PADI4, which subsequently activated TACE. However, RG repressed PADI4 induction, suggesting that PADI4 may be an upstream regulator of TACE.
Inhibition of nuclear factor-κB by red ginseng To further examine whether RG could inhibit inflam- mation, we investigated induction of NF-κB by oxidative stress [24] using a reporter gene assay. We found that exposure to H2O2 significantly increased the NF-κB level
compared to non-oxidative conditions. However, RG pre- treatment significantly repressed NF-κB induction (Fig.
4A). To confirm repression of NF-κB induction by RG, translocation of NF-κB from the cytosol to the nucleus was determined. After exposure to H2O2, the NF-κB level in the nucleus was significantly increased whereas the NF-κB level was significantly decreased by RG pretreat- ment (Fig. 4B, C). These results indicated that transloca- tion of NF-κB to the nucleus as well as NF-κB induction were significantly repressed by RG treatment.
Anti-inflammatory effect of red ginseng
Since NF-κB induction could produce ROS [24], we Fig. 2. Repression of immobilization (IMO)-induced tumor necrosis factor-α converting enzyme (TACE) by red ginseng (RG) in vivo. Mice were stressed by IMO for 30 min and immunohistochemistry analysis using a TACE antibody. PBS, phosphate buffer saline; Control, normal non- stressed control; LPS, lipopolysaccharides-injected group.
Fig. 3. Reversal of peptidyl arginine deiminase type IV (PADI4)-in- duced tumor necrosis factor-α convertase (TACE) expression by red ginseng (RG) in vitro. SK-N-SH cells transfected with siPADI4 were treated with 1 mg/mL RG for 48 h, and exposed to 0.5 mM H2O2 for 2 h. Cells were then lysed, and cell lysates were used for Western blot analysis. Representative data from two experiments are shown.
examined effect of RG on ROS production and NO re- lease to further corroborate repression of NF-κB. When SK-N-SH cells were exposed to H2O2, the level of ROS
increased significantly. However, RG pretreatment sig- nificantly repressed ROS induction (Fig. 5A). To confirm repression of ROS production by RG, LPS-induced NO Fig. 4. Repression of nuclear factor (NF)-κB induction and translocation by red ginseng (RG) in vitro. (A) SK-N-SH cells were treated with 1 mg/
mL of RG and then exposed to 0.1 mM of H2O2 followed by determination of NF-κB activity using a reporter gene assay. (B,C) Increase of NF-κB translocation from cytosol to nucleus by RG. SK-N-SH cells (8×105 cells) were pretreated with 1 mg/mL of RG extract for 48 h, and then washed with phosphate buffer saline (PBS) and exposed to 0.5 mM H2O2 for 0 or 1 h. Cells were then washed with PBS, and nuclear lysates were pre- pared followed by Western blot with p65 antibody. Black and gray bars are non-RG-treated and RG-treated cells, respectively. Control, normal non-stressed control. ***p<0.001.
Fig. 5. Repression of reactive oxygen species (ROS) and nitric oxide (NO) levels by red ginseng (RG) in vitro. (A) SK-N-SH cells were treated with 1 mg/mL of RG for 48 h or with media alone (control). After treatment with 0.5 mM H2O2 for 20 min, the intracellular accumulation of ROS was measured using the fluorescent probe H2DCFDA by flow cytometry. (B) C6 cells were pretreated with 0.5 mg/mL of RG extract for 24 h fol- lowed by the addition of 1 μg/mL of lipopolysaccharides (LPS). The amount of NO2- in cell supernatants was determined. Control, normal non- stressed control; LPS, LPS-treated group. *p<0.05, **p<0.01, and ***p<0.001 vs. without RG or with RG, by ANOVA.
release was examined. When C6 glioma cells were treat- ed with LPS, the NO level increased in a time-dependent manner. Moreover, RG pretreatment significantly inhib- ited the level of release of LPS-induced NO (Fig. 5B) suggesting that RG repressed LPS-induced NO release.
These results demonstrated that RG inhibited the expres- sion of genes involved in inflammation and oxidative stress thereby decreasing production of oxidative com- pounds and subsequently protecting the brains of mice from oxidative damage.
DISCUSSION
IMO stress was previously shown to upregulate the immune response-associated gene TNF-α in the brains of mice [22]. Moreover, restraint stress increases expression of the inducible isoform of NO synthase (iNOS) in rat brains, and iNOS inhibitor aminoguanidine protects from stress-induced pathophysiology [26]. However, how TNF-α expression could be controlled by RG remained unknown. In this study we demonstrated that RG inhib- ited PADI4 via TACE expression followed by inflamma- tion and apoptosis.
Once immune-response associated genes are induced, they could disrupt homeostasis and eventually result in a disease state. Our previous result demonstrated that cell death-associated genes were induced by IMO stress
[22]. Moreover, IMO stress increased apoptosis [27].
Apoptosis is induced by activation of caspases such as caspase-3 [28]. However, in this study we showed that RG repressed caspase-3 expression and increased anti- apoptotic Bcl-2 and p53, indicating that RG repressed apoptosis. RG also repressed TACE expression in vivo (Fig. 2). Moreover, siPADI4 repressed both TACE and PADI4 expression collaterally (Fig. 3), indicating that PADI4 induced TACE expression in the presence of RG.
Consistently, RG could repress NF-κB induction as well as NF-κB translocation under oxidative stress condi- tions (Fig. 4), which subsequently repressed ROS and NO production (Fig. 5). Since restraint stress increases expression of the iNOS in rat brains, and iNOS inhibitor aminoguanidine protects from stress-induced pathophysi- ology [26], our results suggest that RG can modulate gene expression via various activities comprising PADI4, TACE, NF-κB, and ROS followed by a net reduction in cell death.
Physical, psychological or mixed stress in the brain triggers inflammatory responses including the release of several inflammatory cytokines, free radicals, prostanoids and transcription factors, activation of neutrophils, and the release of protein oxidation marker myeloperoxidase culminating in damaged brain cells [29]. In this study, we showed that IMO stress could induce inflammatory responses in the brain, leading to tissue damage. Interest- ingly, anti-inflammatory pathways are also activated in the brain in response to stress, to defend against inflam- mation-induced damage, thus resulting in dualizing pro- and anti-inflammatory responses [29]. RG may thus also induce an anti-inflammatory response to dampen the pro-inflammatory response incurred by oxidative stress.
Further studies will be needed to elucidate the underlying mechanism of our observations.
Transcription in eukaryotic cells can be regulated at the post-translational level by histone modifications in- cluding methylation, phosphorylation, and ubiquitination, and histone arginine methylation is catalyzed by protein arginine methyltransferases [30]. PADI4 demethylates histone arginine at the p21 promoter region [31]. PADI4 thus represses the expression of genes induced by estro- gen and retinoic acid receptors [32,33], serves as a p53 corepressor and represses theexpression of p53 target genes p21/WAF1/CIP1 [31]. Although we demonstrated that siPADI4 attenuated cell death [22] and RG pretreat- ment increased p53 expression (Fig. 1A), we cannot exclude other mechanisms of anti-apoptosis. Our system biology analysis and network analyses showed that IMO stress shows significantly higher induction of cell death- Fig. 6. Red ginseng (RG) inhibits apoptosis in an oxidatively-
stressed brain via tumor necrosis factor (TNF)-α convertase (TACE).
Oxidative stress induces peptidyl arginine deiminase type IV (PADI4), which activates TACE and nuclear factor (NF)-κB translocation, trig- gering cues of inflammation as well as production of reactive oxygen species (ROS). Activation of PADI4 and TACE results in apoptosis.
RG represses TACE via PADI4, thereby inhibiting apoptosis in the brain cells.
associated genes such as HOXA5 (apoptosis), ITGB3BP (killing and adhesion), and LGALS3BP (cell death) than other genes. Moreover, RG pretreatment up-regulates apoptosis-associated genes including caspase, CASP9, and HOXA5, yet RG pretreatment did not result in in- creased cell death. Therefore, RG may also induce other as-yet-unknown inhibitors of apoptosis proteins such as Bruce, cIAP1, cIAP2, Survivin (BIRC5), or XIAP [34-36]
alone or in combination, followed by inhibition of apop- tosis. In this case, RG would show anti-apoptotic activity via caspase-independent pathways. Further studies are warranted to determine which mechanism is involved.
Taken together, we found that oxidative stress by IMO induced PADI4, which activated TACE and subsequently the release of TNF-α and NF-κB activation/translocation, which further induced inflammation and ROS secretion.
In contrast, RG repressed PADI4 expression, thus revers- ing stress-induced gene expression and subsequently inhibiting apoptosis by repressing TACE and NF-κB activation/translocation, and by repressing ROS and NO production (Fig. 6).
ACKNOWLEDGEMENTS
This work was supported by the 2011 grant from the Korean Society of Ginseng and a grant from the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (no. 111035-3).
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